Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome

Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H₂O₂ led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H₂O₂ and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the process.     

津田芜菁MYB77蛋白的原核表达及纯化

目的:建立津田芜菁转录因子MYB77的原核表达系统,并在大肠杆菌中获得表达。方法:RT-PCR获得MYB77的编码序列,将其克隆至pGEM-T载体中,在上下游引物中分别引入HindⅢ和EcoRⅠ酶切位点,PCR获得带酶切位点的目的片段并将其连接到重组表达载体pGEX-KG中,转化大肠杆菌DE3工程菌株,IPTG诱导重组质粒pGEX-KG-MYB77在大肠杆菌DE3中表达带有GST标签的融合蛋白,超声裂解大肠杆菌,用MagneGST ProteinPurification System纯化目的蛋白,通过SDS-PAGE和Western印迹验证GST-MYB77融合蛋白的表达。结果:重组菌株可以表达GST-MYB77融合蛋白,用Western印迹鉴定纯化的融合蛋白,在相对分子质量为55.56×103处检测到目的条带。结论:利用大肠杆菌表达系统获得了较高纯度的GST-MYB77融合蛋白,为进一步研究津田芜菁MYB77蛋白的功能奠定了基础。     

听源性惊厥致P77PMC大鼠杏仁核内胆囊收缩素mRNA短暂性增加

铃声刺激诱发惊厥 ,原位杂交法检测遗传性听源性癫痫易感大鼠 (P77PMC)一次与多次惊厥发作对杏仁核内胆囊收缩素 (CCK)mRNA含量的影响。结果发现 :( 1)惊厥未发作组大鼠杏仁核单位面积内的CCKmRNA阳性神经元数 (No/ 0 0 1mm2 )较少 ,为 8± 1;( 2 )惊厥发作一次组大鼠杏仁核单位面积内CCKmRNA阳性神经元数显著增加 ,发作后 30min时达到峰值 ,为 5 8± 5 (P <0 0 1) ,但是 2h后迅速降为正常 ,为 9± 2 (P >0 0 5 ) ;( 3)惊厥多次发作组大鼠在惊厥发作后 30min时 ,CCKmRNA阳性神经元数亦显著增加 ,为 2 2± 3 (P <0 0 1) ,但明显低于惊厥发作一次组大鼠 (P <0 0 1) ,1h即恢复正常 ,为 9± 3 (P >0 0 5 )。结果表明 ,惊厥发作后P77PMC大鼠杏仁核内CCKmR NA含量呈现迅速而短暂增加的特点 ,表明CCKmRNA参与惊厥的急性发作过程 ,提示CCKmRNA在惊厥发作早期阶段发挥了重要的抗惊厥作用     

The oxidation of endogenous ascorbic acid during histamine secretion by rat peritoneal mast cells

Endogenous ascorbic acid is oxidized to the free radical species by rat mast cells during histamine secretion. This antioxidant may function as a radical scavenger of Superoxide and other membrane-damaging radicals known to be liberated by this process. The high levels of ascorbic acid in mast cells may, therefore, function to protect the cell membrane from oxidative damage and thereby promote cell survival after an extensive secretory response.     

Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos.In this study, flower buds with sizes of 5–7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD₆₀₀) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension.The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD₆₀₀ = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.